HEMATOPOIETIC GROWTH CONTROL AND ONCOGENESIS 
Charles J. Sherr, M.D., Ph.D., Investigator 
Colony-stimulating factor 1 (CSF-1) regulates the 
proliferation, differentiation, and survival of mono- 
nuclear phagocytes. Macrophages deprived of CSF- 1 
undergo growth arrest early in the first gap phase 
(Gj) of the cell cycle and ultimately die. However, 
if transiently starved cells are restimulated with the 
growth factor, they progress synchronously through 
Gi and begin to replicate their chromosomal DNA (S 
phase) 10 h later. CSF-1 stimulation is required 
throughout Gj to ensure cellular DNA synthesis, but 
once cells commit to enter S phase, they can com- 
plete cell division without the growth factor. CSF-1 
binds to membrane-spanning cell surface receptors 
encoded by the FMS proto-oncogene, and the 
ligand-induced activation of the CSF-1 receptor 
(CSF- 1 R) protein-tyrosine kinase (PTK) triggers sig- 
nal transduction through multiple second messen- 
ger pathways. The latter signals act in turn to govern 
the transcription of CSF- 1 -responsive genes, 
thereby determining the magnitude and specificity 
of the biologic response and, ultimately, the deci- 
sion to enter S phase. 
The central goals of Dr. Sherr's laboratory are to 
elucidate mechanisms by which CSF-1 governs pro- 
liferative and differentiative decisions throughout 
the G, interval and to understand how perturbations 
that deregulate elements of CSF- 1 R signal transduc- 
tion pathways contribute to tumorigenesis. 
Independent Mutations in CSF-IR Convert 
It to an Oncoprotein 
Mutations in the CSF- 1 R extracellular domain that 
mimic a conformational change induced by ligand 
binding constitutively induce receptor PTK activity 
and provide sustained signals for cell growth. To 
determine whether multiple sites for such "activat- 
ing mutations" might exist, Dr. Sherr and his col- 
leagues subjected portions of FMS cDNA-encoding 
regions of the receptor extracellular domain to 
chemical mutagenesis and reinserted them into a re- 
troviral expression plasmid to generate libraries of 
FMS genes containing random mutations confined 
to predetermined target cassettes. Transfection of 
these plasmids into fibroblasts yielded morphologi- 
cally transformed cells from which retroviruses 
could be rescued. Amplification by polymerase 
chain reaction of target cassettes from unique inte- 
grated proviruses and nucleotide sequencing re- 
vealed that activating mutations could occur at 
many FMS codons and that certain residues were 
' ' hot spots . ' ' Some of the latter codons in CSF- 1 R are 
conserved in position and context in the evolution- 
arily related receptors for platelet-derived growth 
factor (PDGF) and in the receptor for Steel factor 
(SLF or KIT ligand) , suggesting that analogous mu- 
tations might potentially convert these receptors to 
ligand-independent oncoproteins. 
CSF-IR Signals through Muhiple Pathways 
Ligand binding leads to CSF- IR dimerization, acti- 
vation of receptor PTK activity, and cross-phosphor- 
ylation of aggregated receptor subunits on tyrosine. 
Sites of receptor tyrosine phosphorylation contrib- 
ute to form recognition motifs for certain cytoplas- 
mic effector molecules, whose binding to the re- 
ceptor and/or phosphorylation on tyrosine mediate 
downstream biochemical responses to the growth 
factor. Therefore mutations that selectively elimi- 
nate sites of tyrosine phosphorylation within CSF- 1 R 
can disrupt its interactions with particular effector 
proteins and abrogate signaling through specific 
downstream pathways. Dr. Sheila Shurtleff previ- 
ously found that mutation of one such phosphoryla- 
tion site (Tyr-809) in the cytoplasmic domain of 
CSF- 1 R had no effect on receptor PTK activity, bind- 
ing to phosphatidylinositol 3 -kinase, or the induc- 
tion of several immediate-early response genes, in- 
cluding C-/05, junB, c-jun, or c-ets2, all of which 
encode transcription factors. However, the c-myc 
response to CSF-1 was significantly impaired, and 
cells bearing the mutant receptor were unable to 
proliferate in response to the growth factor. En- 
forced expression of an exogenous c-myc gene res- 
cued the cells' ability to grow in response to CSF-1 . 
These results reveal a bifurcation of CSF-1 signal 
transduction pathways that target fos/jun on the 
one hand and c-myc on the other and demonstrate 
that the latter gene is required for mitogenicity. Dr. 
Sherr and his colleagues are now using cells bearing 
this CSF-IR mutant as a genetic "trap" to screen for 
genes that act downstream of the receptor in control- 
ling the c-myc response. 
CSF-1 Regulates the Expression 
of Gj Cyclins 
Last year, Dr. Hitoshi Matsushime cloned novel 
D-type cyclin genes that are induced by CSF- 1 dur- 
ing the mid-to-late Gj interval. By analogy to B-type 
cyclin, which regulates the activity of the cell divi- 
sion cycle kinase p54'"'''^ to control mitotic entry 
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